FI101159B - Procedure and packaging for the diagnosis of black currant reversal disease - Google Patents

Description

P

ΙΟΊ 159

METHOD AND PACKAGING FOR THE DIAGNOSIS OF BLACK CURRANT VASCULAR DISEASE

The invention relates to a method for diagnosing blackcurrant vascular disease 5 in plants, in particular blackcurrants (Ribes). According to the invention, reverse transcriptase PCR is optionally used in conjunction with the immunocapture method to detect blackcurrant reversion virus (BRV). The invention also relates to a test kit for use in the method. In the work leading to the invention, the RNA of BRV was partially characterized.

From the viral nucleotide sequence thus determined, it was possible to construct primers for use in the method and kit of the invention. In the course of the work leading to the invention, we called the virus Blackcurrant Reversion Associated Virus (BRAV).

Of the approximately 14 different viral diseases or virus-like diseases identified in RiJbes plants, blackcurrant varicose veins are by far the most important blackcurrant crop worldwide (1,2); it also afflicts red currant (3). The disease occurs in Ribes species worldwide ··. with the exception of the Americas. As the name of the disease 25 suggests, it alters the way the plant grows, especially the appearance of the leaf, as a result of which (English name) 'rever-'.! *! Sion 'refers to' restoration to a · primitive primitive • · · * * plant type (3). Diagnosis However, the most reliable • · · * ··· * symptoms of varicose veins appear in flower buds • · · • · «*. * * 30 when they open in early spring.The common European form of varicose veins (E; 1, 2) causes flower buds • · • * · · A significant reduction in hair density and an increase in the color of the buds themselves (3), a more serious form of the disease (R; 1, 2), which is found in Finland (4), Eastern Europe and the countries of the former Soviet Union; distribution, resulting in bracts forming ten instead of five, and further enhancing their coloring (3).

”·: All forms of varicose veins usually cause sterility to flowers, and infected plants quickly become unable to reproduce. In red currant, leaf and flower symptoms are much less noticeable than in black currant; gooseberry has been reported to be immune to 5 infections (3). In nature, the pathogen is spread between plants by blackcurrant mite (Cecidophyopsis ribis Westw.), But not by seeds. Experimentally, it spreads between Ribes plants by stalking (3).

10 The causative agent of varicose veins has not been characterized, despite more than 50 years of research, and the only test for the disease is grafting onto sensitive blackcurrant varieties and waiting for symptoms to develop up to 2 years (3, 5). Such a method of detection is, of course, laborious and uneconomical. Therefore, there is a need for a faster and more reliable method for diagnosing blackcurrant varicose veins.

The present invention describes the isolation of a virus from 20 blackcurrants having similar properties to certain nepoviruses. The virus was transferred from blackcurrant suffering from varicose veins by mechanical inoculation into herbaceous experimental plants. Antiserum was generated for the virus and the nucleotide sequence of the other 3 'end was determined from its two genomic RNAs. Because of this: 1 i it was possible to construct primers for use; j *. in a rapid and specific PCR system for the detection and diagnosis of blackcurrant • · · vascular disease in plants.

.. 30 • · • · ·

One aspect of the invention relates to a method for diagnosing blackcurrant vascular disease in a plant by detecting blackcurrant vascular virus (BRV) therein, comprising: 35 - sampling a test plant, 101159 3 - performing a reverse transcription reaction of a single-stranded cNA: from the viral RNA in the sample using an oligonucleotide primer derived from the viral RNA sequence that is 5 complementary to the nucleotide sequence of the viral RNA, complementary to the first nucleotide sequence fragment of viral RNA, and the second is an oligonucleotide primer derived from said viral RNA corresponding to a second nucleotide sequence fragment of viral RNA different from said first nucleotide sequence. a fragment thereof, and said first and second nucleotide sequence fragments flank the nucleotide sequence to be amplified, and - the amplified product is detected.

20

In another aspect of the invention, said method is combined with an immunocapture method in which a sample is contacted with an antibody of BRV coated with a substrate to separate and concentrate BRV-containing particles from the sample, then. . particles are broken down, e.g. by heating, by the virus • ·. . *. To release RNA.

• · · • · ·

The invention further relates to a diagnostic test kit for diagnosing blackcurrant varicose vein disease in a plant using a reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of blackcurrant varicella virus, BRV, in a plant containing the kit. - for BRV antibody or BRV antibody-coated 35 substrate and 101159 4 - a pair of BRV RNA-derived oligonucleotide primers for RT-PCR to amplify a cDNA fragment complementary to BRV RNA lie.

According to a preferred embodiment, the invention relates to the use of an oligonucleotide primer pair in the method and kit of the invention, so that the primers flank and are capable of amplifying the 3 'proximal 210 bp 10 f fragment of the cDNA formed from viral RNA in a reverse transcription reaction.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows RT-PCR amplification of a 210 bp cDNA-15 fragment of BRV RNA obtained from nucleic acid extracts from the following sources (lane numbers in parentheses): a. Locally and systemically infected BRV-infected Chenopodium quinoa. leaves (1 and 2, respectively); Systemically infected leaves of BRV-infected Nicotiana occidentalis, 37-B (3); healthy C. quinoa leaves (4); and partially purified BRV particles (5). Lane 'M' contains PstI-cleaved λ-DNA as molecular weight markers.

• ·. b. partially purified preparations from the leaves of blackcurrants afflicted with the R- • · · form of varicose veins, obtained from the Agricultural Institute of the Agricultural Research Center (ARC-IH) 30 Piikkiö (2) and Parikkala (3), healthy plants (4) and • · \ purified BRV particles ( 5). Lane 1 contains the buffer control and lane M1 as a 1 kb "ladder" · * ·: molecular weight marker (Gibco BRL).

Figure 2 shows IC-RT-PCR amplification of a 210 bp cDNA fragment of BRV RNA obtained from nucleic acid extracts of plants or mites. In each gel, lane 'M' contains 101159 5 of a 100 bp DNA ladder (Gibco BRL) and lane 'C · contains material from IC-RT-PCR of healthy blackcurrant. The extracts were from the following sources (lane numbers in parentheses): 5 a. Individual blackcurrant shrubs afflicted with the R-form of varicose veins originally obtained from HRS in Piikkiö (1-5) and Parikkala (6-10); and groups of 50 äkämäki mites stalking diseased blackcurrant bushes from the Agriculture Department of the Agricultural Research Center in Piikkiö (11) and Parikkala (12). Zones in lanes 6, 9, and 12 were very weak and do not reproduce well in the image.

b. two blackcurrant shrubs, each with the R-form of swamp-15 nasopharyngeal disease, both of the varieties Lepa black (1, 2), Karila (3, 4), Nikkala (5, 6), Bija (7, 8) and Suont- black (9, 10). Zones in lanes 4 and 9 were very weak and do not reproduce well in the image. No zone was detected in lane 10.

20 c. individual blackcurrant shrubs grown in Kaarina that were either asymptomatic (1) or had symptoms of varicose vein disease form E (2 - 7). The zone in lane 7 was very weak and does not reproduce well in the figure.

25. . Figure 3 shows plant samples from Scotland:. IC-RT-PCR amplification of a 210 bp cDNA fragment of BRV RNA from extracts. In each gel, lanes ‘C’ and ‘M’ represent the analysis of healthy blackcurrant and correspond to a 30-bp DNA ladder (Gibco BRL). The samples are derived from extracts obtained from the following sources • «· * ·] '(lane numbers in brackets): • · • · · a. Healthy plants of blackcurrant varieties Ben Alder (1, 35 7), Ben Lomond (2, 11 ), Ben Nevis (8), Ben Tirran (9) and

Ben Sarek (10) and anonymous varieties with R-form (3, 4) and E-form (5, 6) for varicose veins.

101159 6 b. Chenopodium quinoa plants (1-5) and i? Ibes varieties (6-15). C. quinoa was infected with strawberry latent ring spot virus (1), Arabis mosaic virus (ArMVj - blackcurrant isolate (2) and - lilac isolate (5), 5 raspberry ring spot virus - Scottish isolate (3) and uninfected healthy plant (4). Samples of blackcurrant species are plants previously grafted on jRibes plants with: 10 Yellows disease - varieties Ben Alder (6) and öjebyn (8), infectious variant - variety Öjebyn (10), gooseberry leaf veins Veinbanding disease (GVBD) - variety Ben Nevis with and without GVBD symptoms (11) (9), ArMV - Ben Lomond variety with 15 jaundice symptoms (14) and asymptomatic (15), varicose vein disease R form - varieties Ben More (7) and Amosin Black Blackcurrant (13), healthy Amosin Black Blackcurrant variety (12).

Figure 4 shows IC-RT-PCR amplification of a 210 bp cDNA fragment of BRV RNA from extracts of Silvergier-ters Schwarze from New Zealand. Lane 1, healthy plant; lanes 2 and 3, plants with mild and severe symptoms of varicose veins, respectively; lanes 4 and 5, 25 control samples from healthy and vascular disease, respectively. . blackcurrant from Finland; lane M, 50-; 1000 bp DNA marker (FMC).

• · «• · · • ·

In the work leading to the invention, we have succeeded in isolating .. and probably describing the properties of the newly described virus, blackcurrant vascular virus, BRV.

• · · • · · · '·· In the rooting cuttings of blackcurrant with R-disease, early 35 chlorotic patterns and ring patches developed on the leaves that appeared early. The virus was laboriously mechanically transferred from these symptomatic leaves to the species Chenopodium quinoa Willd. and from this host to other 101159 7 herbaceous test plants. The virus was purified, partially characterized, and anti-serum generated against it. The virus particles were constant in size, about 27 nm in diameter, and sedimented as two nucleo-5 protein components. They contained a Mr 55 kD protein type with a molecular weight that was easily degraded to a 54 kD protein, and two major RNA components, approximately 6700 and 7700 nucleotides in size, each with poly-A tails. Nepoviruses have most of these 10 characteristics, but the virus was not serologically related to the 14 nepoviruses studied or the potential nepovirus.

Specifically, the virus was isolated from a blackcurrant breeding line at the Scottish Crop Research Institute (SCRI). Purified virus was used to generate antiserum by immunizing the rabbit. The resulting antiserum was used to purify and concentrate BRV particles in diagnosable samples.

20

Viral envelope protein and nucleic acid were analyzed.

RNA was isolated and analyzed on an agarose gel, followed by Northern blot analysis. For sequence analysis, cDNA was synthesized from viral RNA to form recombinant clones. DNA was isolated from selected. preliminary restriction analysis of recombinant clones and sec- · ·. . ·. for valence analysis. Thus, it was possible to analyze • · ·. • j *. a sequence containing 1260 3 'terminal nucleotides and a poly-A tail of one RNA component of BRV. Said .. 30 sequence is shown in SEQ. In ID NO: 1.

• l 10 «·· • · · * · * * Sequence analysis allowed the design of primers for use in the method of the invention. Specifically, it allowed the design of primers to amplify a 3'-35 proximal region containing 210 bp of DNA complementary to BRV RNA immediately upstream of the poly-A tail.

101159 8

In a preferred embodiment of the invention, immunocapture reverse transcription PCR (IC-RT-PCR) is used to diagnose blackcurrant varicose veins in plants. In such a method, a sample, such as 5 plant extracts, obtained from suitable symptomatic plant parts, for example leaves, can be used. The BRV particles in these extracts are concentrated and purified by the immunocapture method as described in literature reference (6). A medium such as a PCR tube, a microfuge tube or a microtiter plate coated with antiserum can be used for this purpose. After incubation, viral particles "captured" in antiserum-coated medium are disrupted, for example by heating, to release RNA.

The RNA thus obtained is used to synthesize the first strand of the cDNA in a reverse transcriptase reaction using a reverse transcriptase mixture, for example a commercially available mixture, together with an oligonucleotide primer 20 (primer 1) complementary to the nucleoside immediately upstream of the viral poly-A tail. , and a reverse transcriptase enzyme. After a suitable incubation time, the reverse transcriptase enzyme is inactivated.

The cDNA thus obtained is then amplified in a PCR reaction.

. . ·. According to a preferred embodiment, the 'hot start' method (7) is used for amplification. In the hot start method, DNA * polymerase is activated only after the reaction has reached a high temperature, thus minimizing the amplification of the false target and the formation of unwanted by-products. Hot start can be performed by adding an essential component to the reaction tube only when it has reached a high temperature.

As primers for PCR, the above-mentioned primer 1 is used in combination with another primer, primer 2, which corresponds to the second nucleotide sequence of the virus, which primers 1 and 2 flank the cDNA of the target nucleotide sequence to be amplified.

According to a preferred embodiment of the invention, primer 1 is complementary to the nucleotide sequence of the virus at positions 1-15 upstream of the poly-A tail, and has the sequence 5 'GAAAGGACATTTCAG 3' (SEQ ID NO: 3). Primer 2 corresponds to the nucleotide sequence at positions 199-210 poly-A-tail-10 and is 5 'CGCTGGTGTCTC 3' (SEQ ID NO: 4). Primers can be synthesized according to known principles, for example using the solid phase phosphoramidite method.

The PCR amplification reaction can be performed as an automated process in a thermal cycler, for example, running 30 cycles with template denaturation at 95 ° C for 1 minute, primer base pairing at 37 ° C for 1 minute, and elongation at 72 ° C for 1 minute. The elongation phase is extended after 20 cycles. For detection, PCR products are electrophoresed on an agarose gel, stained with ethidium bromide, and viewed under UV light. Alternatively, PCR reaction products can be fluorescently labeled by initiating their synthesis with a fluorescently labeled primer, whereby the products are detected by automated optical; . ; la detection, using, for example, the ALF Manager System • «. . ·. groups.

• · · • · · • «· • · · tl

The following examples illustrate the invention.

30 • ·

; “Example 1: ISOLATION AND ANALYSIS OF BRV VIRUS

• «· • · ♦ • · · ·’ ·: Plant material, inoculation methods and virus culture.

The blackcurrant material from which the virus was isolated was the breeding line P9 / 5/1 of the Scottish Plant Research Institute (SCRI), which grows on the experimental farm of the Agricultural Research Centre's Orchard (ARC-IH) in Piikkiö, Finland. It 101159 10 was planted as a virus-free material, but it was rapidly infected with the R-form of varicose veins, which was probably derived from infected Finnish blackcurrant shrubs growing nearby.

5 Soft wood cuttings were taken from this plant and rooted in a peat-sand mixture in a greenhouse at the Agricultural Research Centre's Plant Protection Department in Jokioinen. During rooting, new chlorot-10 pigs / yellow patterns and ring patches developed on the new leaves of these cuttings. Such leaves were comminuted in a 2% nicotine solution, pH 9.5, the sap extract was rubbed into the leaves pollinated with carborundum from herbaceous test plants, and the plants were kept in a greenhouse at about 21 ° C. After one such inoculation, symptoms were observed in the leaves of Chenopodium quinoa. The BRV virus found to be contained in this plant was then maintained by serial passages in C. quinoa and other herbaceous hosts. The virus was also stored as infected C. quinoa leaves at 20-20 ° C and -80 ° C.

Virus cleaning. BRV was purified using a slightly modified method described by Frison and Stace-Smith (8) for Arabis mosaic nepovirus. Transplanted and / or systemically infected C. quinoa leaves. . . recovered 15-28 days after inoculation and

. milled with 3 ml / g buffer (0.05 M Na 2 HPO 4, 0.02 M

• · · III ascorbic acid, 0.02 M 2-mercaptoethanol, pH 8.0).

• · φ * The homogenate was filtered through a cheese cloth and clarified by centrifugation at 15,000 g for 20 minutes. The pH of the supernatant liquid was adjusted to 5.0 with dilution, kept overnight at 4 ° C, and centrifuged; was then boiled at 15,000 g for 20 minutes. 1% NaCl and 8% polyethylene glycol (PEG 6000) were added to the supernatant liquid, and the mixture was stirred at 4 ° C for 1 hour, followed by centrifugation at 15,000 g for 20 minutes. The pellet was resuspended in 101159 11 tenths of the original volume of 0.05 M Na-citrate buffer, pH 7, containing 1% NaCl, and the mixture was stirred for 1 hour before being centrifuged again at 15,000 g for 20 minutes. Supernatant liquid 5 was collected and stored, and the pellet was redissolved from half its original volume in 0.05 M Na-citrate buffer before centrifuging again at 15,000 g for 20 minutes. The supernatant liquid was again collected and combined with the previously obtained supernatant sample, followed by centrifugation at 105,000 g for 90 minutes. Virus-containing pellets were resuspended in 0.05 M citrate buffer. In other experiments, BRV was purified from Nicotiana benthalmiana Do-min. clarifying plant extracts with 50% (v / v) chloro-15 form. The aqueous phase was further clarified and concentrated in one round of high and low speed centrifugation. Virus preparations were further purified by placing 100-500 μΐ of final virus suspension on 10-40% sucrose density gradients in 0.05 M citrate buffer prepared in Beckmann SW41 or SW50.1 tubes.

Sucrose density gradient tubes were centrifuged at 38,000 rpm for 90 minutes (SW41) or 45,000 rpm for 50 minutes (SW50.1), and the gradients were fractionated by upward displacement using an ISCO density gradient fractionator, absorbance was monitored at 254 nm. . Virus-containing fractions are combined. .1 2 3. was diluted with an equal volume of citrate buffer and centrifuged at 180,000 g for 2 hours.

The final pellets were resuspended in a small amount of citrate buffer.

• · »« l 2 • · «3 1 Virus yields were relatively low, although preparations were made from BRV-infected herbaceous plants at the optimal time of year for oi expression.

35 Maximum yields were approximately 4-5 mg / kg leaf. Purified virus preparations, after sucrose density gradient centrifugation, usually formed two closely related 101159 12 sedimentation zones of light scattering identified from the two major absorbance peaks in the middle stages of the gradient. Fractions of these peaks were associated with the highest infectivity.

5

Electron microscopy. Purified virus preparations were placed on carbon-coated copper grids and stained negatively and examined with a JEOL JEM-100SX or JEOL 100S electron microscope.

10

Under an electron microscope, the virus preparations were found to contain many standard-sized particles, some of which were angular in outline (icosahedrons) and measured to a diameter of about 27 nm. All of the four negative dyes used permeated several particles, but the proportion of particles permeated in the dye appeared to be higher in ammonium molybdate, pH 7.0, than in PTA (phosphotungstic acid), pH 7.0, methionine tungstate, pH 7.0, or uranyl acetate. , pH 3.5. Particle-20 particles, which were partially permeated in color in uranyl acetate, appeared to have either a central core or a thicker protein shell that was not present in the particles in other dyes. Although these differences in particle appearance may be artificial products provided by the uranyl acetate dye, they have not previously been observed for certain nepovirus particles in this Dye.

· T • · 4 0 4 • · ·

Analysis of viral envelope protein and nucleic acid.

* Viral envelope protein size was determined by electrophoresis on 10% SDS-PAGE gels as described by Laemmli (9). Nucleic acid was isolated from purified • · * *. * * Virus preparations either using a commercial Micro-Fast- * * ·: Track kit (Invitrogen) to isolate RNA with a poly-A tail, or by extraction with phenol, phenol-chloro-35 form, and by doping with ethanol. The extracted nucleic acid was denatured with glyoxal and dimethyl sulfoxide-101'15913a, analyzed on 1% agarose gels, and stained with ethidium bromide as described by Sambrook et al. (10).

For Northern blot analysis, RNAs were transferred from aga-5 rose gels to a Hybond-N membrane (Amersham) and the membrane was fixed with UV and glyoxal was removed by heating at 80 ° C for 2 hours. The spots were examined with viral cDNA labeled with digoxigenin-dUTP (Boehringer Mannheim). Hybridization reactions were detected using 10 kits of DIG Luminescent Detection Kit (Boehringer Mannheim).

When analyzed on SDS-PAGE gels, protein extracts of purified viral preparations usually migrated into two distinct bands of approximately 54 kD and 55 kD. However, in some preparations, only a 54 kD viral protein band was detected. When such a viral preparation from which only this protein was obtained was inoculated into C. quinoa, it produced symptoms similar to the initial BRV infection 20, and the virus particle preparation purified from these infected plants contained both 54 kD and 55 kD proteins. This suggests that both proteins are derived from the virus, and that the smaller appears probably as a result of the degradation of the larger protein. These two protein bands were detected. . western blot of similarly purified BRV preparations • ·. assays when analyzed with BRV polyclonal antiserum.

• · c .. 30 When denaturing agarose gels were analyzed, viral nucleic acid preparations produced two major bands.

• (i * · ['The nucleic acid profiles obtained from the samples were identical to those obtained either by absorption into a commercial oligo-dT cellulose matrix (Invitrogen) or by phenol extraction and ethanol precipitation. When 10 Mg / ml RNase A was added to the preparations. , no further nucleic acids were detected, suggesting that the viral nucleic acid was 101159 14 RNAs, and absorption of RNA on oligo-dT indicates that two RNA species have been polyadenylated using the MCID image analysis system (version 1.2 ) and a 0.24-9.5 kb RNA ladder (Gibco BRL) as a molecular size marker, the size of the two RNA-5 molecules was estimated to be approximately 6700 and 7700 nucleotides, respectively.

cDNA cloning and sequence analysis. First strand cDNA was synthesized from viral RNA with oligo-dT primers 10 using the 'First Strand cDNA Synthesis Kit' (Pharmacia). The reaction mixture was immediately used for the synthesis of the second strand according to Sambrook et al. (10). NotI linkers were added to the cDNA, after which the cDNA was ligated into the NotI digested, dephosphorylated Bluescript SK + vector and transformed by electroporation into JM109 cells. DNA

were isolated from selected recombinant clones by alkaline lysis (10) either on a miniature scale for preliminary restriction analysis or on a large scale for sequence analysis. Sequence analysis was performed with 20 automated sequencing using the ALF Manager system, version 2.5. Sequence data were analyzed with various programs using the Genetics Computer Group (GCG) sequence analysis software package, version 8.0.

Antiserum was prepared by subcutaneous immunization of rabbits. . about 50-100 μg of purified virus in Freund's incomplete adjuvant. Two booster injections were given intramuscularly every 14 days. Serum was taken from the animal 14 days after the last injection and stored at 30-30 ° C.

• · • · · ·· »

"· * * Example 2 DIAGNOSIS OF BLACK CURRANT VASCULAR DISEASE

· * ·: IN PLANTS

35 Design of oligonucleotide primers. Based on the sequence of the 3 'end of the BRV RNA as defined above, the following oligonucleotides were synthesized: 5' GAAAGGACATTTCAG 3 '(primer 101159 15 1) complementary to the viral nucleotide sequence at positions 1-15 upstream of the poly-A tail, and 5 'CGCTGGTGTCTC 3' (primer 2), which corresponds to the viral nucleotide sequence at positions 199-210 upstream of the poly-A tail.

The 230 bp fraction of the viral nucleotide sequence converted to DNA, including the poly-A tail, is shown in the attached SEQ. In ID NO: 2. The primers were synthesized by the solid phase phosphoramidite method on a 0.01 micromolar scale and the salts were removed before use.

Extraction of viral RNA from purified virus particles, infected plants and ticks. An equal volume of RNA extraction buffer (0.1 M glycine, 0.1 M Tris, pH 8.6, 0.1 M NaCl, 0.01 mL) was added to the suspension of BRV particles purified as described above. M EDTA, 0.2% SDS (sodium dodecyl sulfate), 0.2% sodium dodecyl sarcosine). RNA was extracted from this suspension by adding an equal volume of phenol / chloro-20 Form, and RNA was precipitated from the aqueous phase with 1/10 volume of 3 M NaOAc, pH 5.2, and 2.5 volumes of ethanol.

The pellet containing RNA was resuspended in 8 water and used directly for the synthesis of the first strand of cDNA.

25. . . To extract nucleic acid from BRV-infected herbaceous plants, the symptomatic leaves were ground in a liquid wash and ground with a pestle and mortar in RNA extraction buffer (2 ml / g leaf). The homogenate was extracted with an equal volume of phenol and phenol / chloroform and the nucleic acid was ethanol precipitated as described above. To extract nucleic acid from Ribes leaves, • · about 300 μg of young leaves were ground in a liquid nitrogen Eppen-Dorf tube with a glass rod. 500 μ TE buffer (10 mM Tris, 1 mM EDTA), pH 7.4, was added to the extract with stirring. The homogenate was frozen for 20 minutes at -20 ° C, thawed at room temperature, and extracted with 0.2119 by volume of 0.25 volumes of chloroform for 5 minutes. The emulsion was centrifuged at 2000 rpm for 5 minutes, the aqueous phase was removed and centrifuged at 12,000 rpm for 10 minutes. The pellet was resuspended in 500 μl of RNA extraction buffer, extracted twice with phenol / chloroform, and the nucleic acid was precipitated with ethanol at -70 ° C.

To extract the nucleic acid from the stinging mites, the budding blackcurrant buds from the 10 shrubs plagued by varicose veins R-form growing in the Jokioinen field of the Plant Protection Plant were cut in half and the leaf primordia was gently shaken from the tissues in a small amount of water. From this punk suspension, groups of about 50 mites were picked with 15 thin hairs in Eppendorf tubes and centrifuged rapidly to pellet the mites. The tick-containing pellet was washed once in water and the ticks were re-pelleted by rapid centrifugation. The pellet-containing pellets were ground by adding liquid nitrogen and then triturated with a glass rod in 500 μ 500ιββΆ RNA extraction buffer before phenol / chloroform extraction and ethanol precipitation of the nucleic acid as described above.

Plant samples for analysis. Plant samples were collected for 25 PCR analyzes in late spring / early summer. In Finland !! '! the samples were from field plants of the Jokioinen Plant Protection Institute (IPP) with symptoms of varicose veins • · · * · ”* in flowers and / or leaves, from the collections of germplasm of the Agricultural Research Center, • ·« * · * * and 30. from private gardens in southern Finland.

• · • * ·· Healthy plants (certified virus-free plants) were »·· ::: originally from the Laukaa Research, ·’ ♦ and Elite Plant Unit of the Agricultural Research Center and were subsequently maintained in sheltered greenhouses at IPP, Jokioinen.

35 In Scotland, samples were obtained from certified virus-free mother plants kept in a screen house, SCRI, Dundee, and were regularly inspected to ensure that they were at all times free from all known viral diseases of Ribes and from these and other such plants. , previously infected by stemming with known 5 viruses or virus-like factors, or infected with Eriophyidae mite mites derived from blackcurrant shrubs suffering from blackcurrant varicose veins. Samples from New Zealand were from both healthy and varicose veins blackhe-10 prayers, the variety Silvergierters Schwarze. Varicose vein disease samples had either mild or severe symptoms caused by Form E disease. Nepovirus samples in C. quinoa were from cultures maintained in SCRI.

15 First strand cDNA synthesis. For the synthesis of the first strand of cDNA, RNA was either obtained directly from purified BRV particles or extracted from BRV particles recovered from plant or mite extracts by immunocapture. In a synthesis starting from RNA isolated directly from purified particles, the reaction mixture contained 8 μΐ RNA in water, 1 μΐ primer 1, 1 μΐ 200 mM DTT, and 5 μΐ reverse transcription (RT) mixture. The RT mixture was either a 'First Strand Synthesis Kit' (Pharmacia Biotech) or a mixture containing 135 mM Tris-HCl, pH 8, 200 mM KCl, 30 mM MgCl 2, 30 mM DTT: (dithiothreitol), 5.5 mM each dNTP, 240 • · * * mg / ml RNase / DNase-free BSA (Pharmacia Biotech), • ♦ · ** "* 2.7 U / μΙ RNAguard- (Pharmacia Biotech) and 3 • · r U / μΙ mouse reverse transcriptase (Pharmacia Biotech).

Reactions were incubated at 37 ° C for 1 hour and then at 90 ° C for 5 minutes to inactivate reverse transcriptase.

• ·

Immunokaappaus method. Immunocapture of BRV particles from 35 leaf and mite extracts largely followed the method of Nolasco et al. (6) with minor modifications. The leaves (0.3 g) were triturated in liquid nitrogen and extracted from a bed and mortar 101159 18 using 2.1 ml of cold TE buffer, pH 8.0. About 0.5 ml of the extract was transferred to an Eppendorf tube, frozen at -20 ° C for 20 minutes, thawed and centrifuged again for 5 minutes at 3000 rpm at 4 ° C. An aliquot (50 μΐ) of the clear supernatant was then placed in PCR tubes previously incubated with 50 μl of a 1: 150 dilution of viral antiserum in 50 mM sodium carbonate, pH 9.6, at 37 ° C for 2 hours, followed by it was washed twice with 100 PBS (phosphate buffered saline-10 solution) containing 0.05% Tween 20 (PBS-Tween). After the clear plant extract was added to the coated PCR tubes, they were incubated at 37 ° C for 4 hours or at 7 ° C overnight. After incubation, the PCR tubes were emptied, washed twice with 100 PBS-Tween, and 8 μΐ water was added and the tubes were heated at 65 ° C for 10 minutes to decompose the "captured" virus particles. The tubes were transferred to ice and used directly for first strand synthesis as described above.

20 PCR. The "hot start" method was used (7). The PCR reaction components were pipetted into PCR tubes, after which cDNA samples were added. The reaction mixture (50 μΐ) contained 20-40 pmol of each primer and 400 μΜ dNTP and 1 x DyNAzyme ™ DNA polymerase buffer. The reaction mixture was covered with molten DyNAWaxT® (Finnzymes), which solidifies rapidly upon contact with the reaction mixture. To this wax layer was added 50 μΐ of a solution containing 2 U of DyNAzyme NA DNA polymerase (Finnzymes) and 5 μΐ of the first strand cDNA reaction in 1x DyNAzyme ™ polymerase buffer.

30 RT-PCR amplifications were performed on a Hybaid thermal cycler (Omni- • · · gene) running for 30 cycles: template denaturation • · · '· / ·' at 95 ° C for 1 minute, primer base pairing at 37 ° C 1 · '· minute and extension at 72 ° C for 1 minute. 30 cycles

then the products were extended at 72 ° C for 10 minutes 35 and cooled to 4 ° C. Aliquots of PCR reactions (30 μΐ) were electrophoresed on 1.5% or 2% agarose gels in TBE buffer (0.045 M Tris-borate, 1 mM

101159 19 EDTA) and the gels were stained with ethidium bromide and viewed under UV light.

The results of the PCR reactions are shown in Figures 1-4. It can be seen that the use of such primers from the cloned cDNA provides reliable amplification of the product by the "hot start" method. Therefore, the "hot start" method was used in all these studies and successfully amplified the product of the expected size when si-10 was used with RNA obtained from purified BRV particles (Figures 1a and Ib, lane 5), BRV infection c. quinoa (lanes 1 and 2) and N. occidentalis (lane 3) (Fig. 1a). After BRV particles from infected Ribes leaves were first concentrated and partially purified, as described, a 210 bp product was obtained from samples of blackcurrant lysate with varicose veins from Piikkiö (lane 2, Figure IB) and Parikkala (lane 3, Figure Ib). In Figures 1a and Ib, buffer containers are run in lane 1 and healthy plant samples in lane 4.

20

A more reliable and efficient 210 bp PCR product was detected in Ribes leaves throughout the period using IC-RT-PCR. A specific product was found in the leaves of 10 field blackcurrants afflicted with the R-form of swamp-25 nasopharyngeal disease and were originally obtained from Agriculture. . . from the Horticultural Institute of Piikkiö and [Parikkala (Fig. 2a, lanes 1-10) as well as from groups of 50 mites • · «(Cecidophyopsis ribis) picked from the buds of these • ·« • · · * blackcurrants (Fig. 2a, 30 lanes 11 -12). However, in some of these assays: ** very little 210 bp PCR product was detected (Fig. • · «* 2a, lanes 5, 6, 9, and 12).

• ·

Linking infection to BRVzhen and blackcurrant varicose veins-35 disease. In attempts to determine the association of varicose veins with the detection of this 210 bp PCR product, IC-1: RT-PCR was used on leaves from a variety of both 101159 20 varicose veins and apparently healthy blackcurrants. When examining the Finnish material, the PCR product was found from leaf samples from: 5 Bija, Karila, Lepa black, Nikkala and 5 Suont-black shrubs from each of the two shrubs growing in the HRS field in Piikkiö and having varicose vein form R and flower symptoms (Figure 2b, lanes 1-10), and six blackcurrant shrubs growing in private 10 gardens near Kaarina in Finland with leaf and flower symptoms typical of varicose vein disease form E (Figure 2c, lanes 2-7). However, the intensity of the stained PCR product varied between samples from each plant as well as between samples taken from the branches of the same plant, suggesting that the virus concentration and / or its distribution in plants may be uneven (Figure 2b, c). No zone of 210 bp was found in plant assays performed on plants from gardens that did not show symptoms of varicose veins (Fig. 2c, lane 1).

In further studies, IC-RT-PCR analysis was performed on plants growing in the SCRI blackcurrant test field at the Plant Protection Institute (IPP) in Jokioinen, which had been established for 25 varicella pathogens and its acacia tick vector. . . to determine the spread. The studied plants were planted as • «] m'm as tick-free and virus-free cuttings between infectious mucus shrubs plagued by weevil mites * · * and having leaf and flower veins of R-form of varicose veins.

• · • · • * · • · ·: All the plants studied were infected with acacia mites and ♦ · showed varying degrees of leaf and flower symptoms typical of the R-form of varicose veins. In each of the random leaf samples taken from each of the 5 plants of Neosyp and Ben 35 Tirran, a 210 bp product was found in all but 2 of the 10 samples showing symptoms of R-form 101159 21, although the intensity of the product varied between plants of each variety. (table 1). Other assays were performed on random leaf samples from blackcurrants (unregistered varieties) from 28 private gardens in different regions of southern Finland where E-form of the disease is prevalent. The 210 bp band was found in 11 of 15 samples with clear symptoms of varicose vein form E, but not in 13 of I4 plants without obvious symptoms of varicose veins (Table 1). A very small amount of product was found in one plant that was apparently free of symptoms of varicose veins (Table 1), but it is possible that this was due to a recent infection with the pathogen at which the symptoms had not yet developed.

15 TABLE 1 Results of IC-RT-PCR for the detection of BRV in different blackcurrant material, some with symptoms of varicose veins and some with no. Blackcurrants grew in fields in different regions-20 Sat in Finland.

Sample no. Location Symptoms PCR results I ΊΡΡ S + 25 2 * IPP S ++

·. . 3 ΊΡΡ S

• · * 4 ΊΡΡ S ++ • · · «· · ::: 5 * ipp s ++ • # · 6 * IPP M +

30 7 ΊΡΡ M

• ·: 8 * IPP M ++ «· · V; 9 * IPP M + • · 10 ΊΡΡ s + II Forssa S +

35 12 Forssa O

13 Forssa 0 14 Forssa 0 101159 22 15 Forssa O -

16 Forssa O

17 Forssa O - 18 Forssa S ++

5 19 Forssa M

20 Forssa O

21 Forssa O -

22 Forssa M

23 Forssa O

10 24 Forssa M

25 Forssa O

26 Sorro O (+) 27 Sorro S ++ 2 8 Kustavi S ++ 15 29 Kustavi M (+) 30 Turku S ++ 31 Turku S ++

3 2 Turku S

3 3 Turku O

20 34 Turku S ++ 3 5 Turku S ++ 36 Turku S ++ 37 Turku S ++ 38 Nokia O - 25 _ • · t • Ψ [l Samples 1-5 were of the Neosyp variety and 6-10 of Ben Tirran • · «'** variety that grew on the experimental field of the Finnish Plant Protection Agency (IPP) • · ·' · * * in Jokioinen. The plants had the R-30 form of varicose veins. Samples 11-38 were unregistered varieties • ·! '·· from areas where varicose vein form E was prevalent.

»« »• · · • · · • O = No symptoms; S = severe symptoms; M = mild symptoms.

- = PCR product not detected; (+) = weak PCR product detected; + = PCR product detected; ++ = strong PCR product detected.

101159 23

Samples for IC-RT-PCR from Scotland were from laboratory cultures of Ribes plants that were either infected or uninfected with the vascular pathogen, or from Ribes or Chenopodium quinoa, which had known diseases such as Ribes virus or viral disease. In these studies, a specific 210 bp PCR product was present only in the assays of plants known to be infected with the causative agent of vascular disease (R and E forms) (Figure 3a, lanes 3, 4, 5 and 6 and 10 Figure 3b, lanes 7 and 13). The product was not present in virus-tested healthy plants from six blackcurrant cultivars (Fig. 3a, lanes 1, 2, 7, 8, 9, 10 and 11 and Fig. 3b, lane 12) or in blackcurrants naturally infected or stemmed with Arabis mosaic-15. povirus (ArMV; Fig. 3b, lanes 14 and 15), or Yellows (lanes 6 and 8), infectious variant (lane 10), or vein banding of gooseberry (lanes 9 and 11). The 210 bp product 20 was also not detected after IC-RT-PCR of nucleic acid extracts from C. guinoa plants infected with either ArMV (2 isolates, lanes 2 and 5), raspberry ring patch ( Scottish strain, lane 3) or strawberry latent ring-spot (Scottish-25 strain) nepoviruses (lane 1) (Fig. 3b). The product of 210 ... bp was also found in studies performed • ·. . ·. with three Ben Nevis blackcurrant plants and two plants • • «« ϊ · \ wool of both varieties Ben Lomond and Ben More, which had been infected 4 years earlier by udder mites 30 from a blackcurrant shrub with vascular • · • '* symptoms of disease E, and which was grown in a heated • · · *. * * greenhouse. However, only three Ben Nevis · * - r plants had clear symptoms of varicose veins, but these .... did not appear until the fourth year after tick infestation.

35 Undoubtedly, therefore, the mite spread BRV to all 7 plants. Ticks also spread the causative agent of varicose veins * to at least Ben Nevis plants. The absence of varicose vein symptoms in the other two varieties may be due to a known delay in the onset of varicose veins after inoculation (3), and this delay was confirmed by the small amount of inoculum used (mites compared to 5 grafting inoculations) and warm conditions.

IC-RT-PCR also revealed a 210 bp product in all 10 blackcurrant samples of Newcastle disease from New Zealand, whether with mild or severe symptoms (Figure 4, lanes 2 and 3), but no product was detected in healthy plants ( Figure 4, lanes 1 and 4).

Lane 5 in Figure 4 shows a positive control sample of 15 blackcurrants infected with varicose veins from Finland.

• · • · · • · · · · • · · • · • • • • • • •

• M

• · · • · · · • · • · 101159 25

REFERENCES

1. Jones, A. T. 1993. Defining the problem: Reversion disease and eriophyid mite vectors in Europe. Proceedings 5 of the Risk Assessment Workshop, 17-18 August 1992> Corvallis, Oregon, USA, pp. 1-4.

2. Jones, A. T. 1995. Recent developments affecting the control of two important diseases of small fruit crops in 10 the UK. Acta Hortic. 385: 64-69.

3. Adams, A. N., and Thresh, J. M. 1987. Reversion of Blackcurrant. Pages 133-136 in: Virus Diseases of Small Fruit-s. R. H. Converse, ed. USDA Agriculture Handbook No. 631.

15 4. Bremer, K., and Heikinheimo, O. 1980. Problems of the reversion disease of Ribes in Finland. Acta Hortic. 95: 87-91.

20 5. Jones, A. T. 1992. Appendix I. Recommended indexing procedures for detecting viruses, viroids, mycoplasma- and Rickettsia-like organisms and virus-like diseases in small fruit crops: Currants and Gooseberries (Ribes). Acta Hortic. 308: 51-154.

25. . 6. Nolasco, G. de Bias, C., Torres, V., and Ponz, F. 1993.

·. · ,! A method of combining immunocapture and PCR amplification in: T: a microtitre plate for detection of plant viruses and subviral pathogens. J. Virol. Methods 45: 201-218.

·. 30 • · · 7. Erlich, H. A., Gelfand, D., and Sninsky, J. J. 1991.

• t · * · Recent Advances in the polymerase chain reaction. Science: 252: 1643-1651.

35 8. Frison, E. A., and Stace-Smith, R. 1992. Cross-reacting and heterospecific Monoclonal antibodies produced against Arabis mosaic virus. J. Gen. Virol. 73: 2525-2530.

101159 26 9. Laeitimli, U. K. 1970. Cleavage of structural Proteins during assembly of the head of bacteriophage T4. Nature (London) 227: 680-685.

5 10. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989).

Molecular Cloning. A laboratory Manual, 2nd edn. New York: Cold Spring Harbor Laboratory.

10 • · • · · * · · • · · · · »» • 1 · • · • t • ·.

• «· • · · • · · · • · 101159 27

Sequence Listing

(1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Oy AboaTech Ab

(B) STREET: Tykistökatu 4 A

(C) CITY: Turku (E) COUNTRY: Finland (F) POSTAL NUMBER (ZIP): 20520 (ii) NAME OF THE INVENTION: Method and Package for Diagnosis of Blackcurrant Vascular Disease (iii) NUMBER OF SEQUENCES: 4 (iv) MACHINE CODE: (A) Floppy disk (B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patent Publication # 1.0, Version # 1.25 (EPO) (2) SEQUENCE ID NO: 1 INFORMATION: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1280 base pairs (B) TYPE: nucleotide (C) STRENGTH: single-stranded (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: genomic RNA transformed into DNA (vi) ORIGIN: (A) ORGANISM: BRV (Blackcurrant reversion virus) • ·: (ix) characteristic: II! (A) NAME / explanation: other feature: (B) LOCATION: 168..170 (D) OTHER INFORMATION: / note = "TAC stop codon of the putative ORF at the 5 'end" • «• · <« «· V ; (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 • · • · · TTGGCTATGG CCACGATGTG AATCATGGAG TAACGCACCA TGAGTCCCCC GATAATTCGG 60 TCCCAAGAGA CTACACTTGG GTTGAAAATG ATGGTAAACC ATCCGTATGG GTCTATGCTC 120 ATATGTTAAG AGTTGTCTTA CTGTATATGG CTTCTACATA AGGTCGGTAC CCCTGAAATA 180 GGGATCCTGT TCGCTTGCGT GGTTGTAGGA GTGCTAAGTA TAGTTGCAAC ATAAATATGA 240 28 101 159 CTTGGGAGAC AAAGTCTCAT CCACCTTGGA ACCGCACAGG GATATAACTC AAAGCGGAAT 300 GGTAGTAAAC TACTTGCCCT ATCAGTAGGG ATGGGAACGC TCATTATTAG CAAACCCTTA 360 CCCTGAACAT TAGGTGCTTC AGCAATATGA GGCGAAAGAC TAGGGAGAAA GTTCCTATGG 420 ACGTGTAATT CGTCCACTCG AGATGGGAGT CATGCCATCG TGTTATCTTA TTTTCTTGTA 480 TTGTATTTCA ATTTCAAAGA AAGTTGCTGG ATCTAAACCA GACCCAGGTG AGTGGACCTT 540 CTGCGTCCAC TCGGTTTGGT ATTTTACGAT TAAATACCCA GGTCTACTGC TTCCGAGCCT 600 GATAAAATCT TAAAGCGCCA TCGTTCAGTA CTTGCTGGCA TGGTGACCTG AGTGATGATG 660 CTTTATGAAT CATCCAATAG TTTCTTTGTT TCTATTTCTG GGAAATCCTT TTGGAGAGTG 720 CCCATGAGAA TAATGAAGCG TTTAGTACAC GATATAGTAC TATCTCTGTT CCAGGAGATT 780 GTGCCGGAAA TATAGTCGA T CATTTTATGA TCAAGGTATT TCTGGATGGG CTCGTTGCGA 840 AGAGTTTTCG CTCAAGGTTC ACATGACTGT AAATTGTAAG TGTACTCAAG ATGTTTCTAG 900 TCTAGGCAAC TAGATGCATT GGATGAGGTC CCGATTGATA CGGGGGGATA ATTAATCCCA 960 GCTTACGGTG TGTGCCGACT TATATGGAAG TGTCCATATC GCATTTTGCT TGTTTTTAGA 1020 CATAAATAAG CACTCTTTAC GCGTGTAATA CGCTGGTGTC TCATTATATA CTAGCGTTGC 1080 AGCGCTAGGA CTACCTAAGA CAACCGTATT ATACGTTTGC TTTATTTATT TTTGTTACTC 1140 CTGTTTAGCA GGTCGTGCCT TCAGTAAGCA CACAAAAATA TTTTCCATTT TTAGGTTTTG 1200 GTTAAGAGTC ATTTTATTGA GCACTTTTCT TTTAGAGAAT AGAGTCTGAA ATGTCCTTTC 1260 AAAAAAAAAA AAAAAAAAAA 1280 (2) SEQUENCE ID NO: 2 INFORMATION: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 230 base pairs (B) TYPE (s) linear • · • · · (ii) MOLECULAR TYPE: genomic RNA converted to DNA • · ·. · *. (Vi) ORIGIN: (A) ORGANISM: BRV (Blackcurrant reversion virus) "(Xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2 101 159 29 CGCTGGTGTC TCATTATATA CTAGCGTTGC AGCGCTAGGA CTACCTAAGA CAACCGTATT 60 ATACGTTTGC TTTATTTATT TTTGTTACTC CTGTTTAGCA GGTCGTGCCT TCAGTAAGCA 120 CACAAAAATA TTTTCCATTT TTAGGTTTTG GTTAAGAGTC ATTTTATTGA GCACTTTTCT 180 TTTAGAGAAT AGAGTCTGAA ATGTCCTTTC AAAAAAAAAA AAAAAAAAAA 230 (2) SEQUENCE ID NO: 3 INFORMATION: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 base pairs (B) single type (B) TYPE ( (ii) MOLECULAR TYPE: DNA (synthetic) (ix) CHARACTERISTIC: (A) NAME / EXPLANATION: other feature (B) LOCATION: 1..15 (D) OTHER INFORMATION: / note = "primer 1» (xi) SEQUENCE DESCRIPTION : SEQUENCE ID NO: 3: GAAAGGACAT TTCAG 15 (2) SEQUENCE ID NO: 4 INFORMATION: (i) SEQUENCE CHARACTERISTICS: *: (A) LENGTH: 12 base pairs (B) TYPE: nucleotide (C) STRENGTH * single stranded 1 1 (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (synthesis etinen) • · • «o» · • · · '·) 1 (ix) CHARACTERISTIC:. [: (A) NAME / EXPLANATION: other feature' “(B) LOCATION: 1..12 (D) OTHER INFORMATION : / note = "primer 2" (Xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO: 4: CGCTGGTGTC TC 12

Claims (14)

101159 A method for diagnosing blackcurrant vascular disease in a plant by detecting Blackcurrant Reversion Virus (BRV), which method comprises the steps of: - sampling a test plant, - performing a reverse transcription reaction on a sample to produce single-stranded cDNA in a sample From the RNA using an oligonucleotide primer derived from the viral RNA sequence that is complementary to the nucleotide sequence of the viral RNA, - the cDNA is amplified by polymerase chain reaction using a pair of oligonucleotide primers, one of which is a viral RNA sequence the first nucleotide sequence fragment of viral RNA, and the second is an oligonucleotide primer derived from said viral RNA corresponding to a second nucleotide sequence fragment of viral RNA different from said first nucleotide sequence fragment. and said first and second nucleotide sequence fragments flank the amplifiable nucleotide sequence, and. - the amplified product is identified. • «· ♦ ♦ · •« · • · · • t A method according to claim 1, characterized in that the sample is an extract obtained from a plant. • t Method according to claim 1, characterized in that the sample contains BRV particles. A method according to claim 1 or 2, characterized in that prior to the reverse transcription reaction, an immunocapture is performed in which the sample is contacted with a BRV antibody immobilized on a support, after which the trapped viral particles are disrupted. A method according to claim 1, characterized in that electrophoresis is used to detect the amplified product. Process according to Claim 1, characterized in that the polymerase chain reaction is carried out by the Mhot-start method. 7. Diagnostic test kit for diagnosing blackcurrant varicose veins in plants using reverse transcriptase polymerase chain reaction (RT-PCR) for detection of Blackcurrant Reversion Virus (BRV) in a plant comprising - an antibody to BRV antibody or BRV for substrate and RT-PCR, a pair of oligo-20 nucleotide primers derived from BRV RNA to amplify a cDNA fragment complementary to BRV RNA. The kit of claim 7, characterized in that the oligonucleotide primers are designed to amplify a DNA fragment that is complementary to vi. . to the 3 ′ proximal 210 bp fragment of rus RNA. • · · • · · • « The kit of claim 8, characterized in that the 3 'proximal 210 bp fragment has the nucleotide sequence 1-210 described in SEQ. ID. N0: 2. * · * «•« · • · «• · 10. An oligonucleotide primer for amplifying a cDNA fragment complementary to a BRV (Blackcurrant Reversion Virus) RNA fragment. 35 i <<>; The oligonucleotide primer of claim 10 for amplifying a cDNA fragment that is complementary to a 3 'proximal 210 bp fragment of viral RNA. The oligonucleotide primer of claim 11, having the DNA sequence 5 'GAAAGGACATTTCAG 3', The oligonucleotide primer of claim 11, having the DNA sequence 5 · CGCTGGTGTCTC 3 '. 14. Antibody produced for Blackcurrant Reversion Virus (BRV). 15